Wajih Hammami; Olfa Kilani; Mariem Ben Khelifa; Wiem Ayed; Abderrezzak Bouzouita; Fethi Zhioua; Sonia Abdelhak; Ahlem Amouri

Research Article

Austin J Reprod Med Infertil. 2015;2(2): 1012.

Genetic Diagnosis in Non-Obstructive Azoospermic Tunisian Men

Wajih Hammami^1,2, Olfa Kilani^1,3, Mariem Ben Khelifa^1,2, Wiem Ayed^1,3, Abderrezzak Bouzouita^3,5, Fethi Zhioua^3,4, Sonia Abdelhak² and Ahlem Amouri^1,2,3*

¹Department of Histology and Cytogenetics, Institut Pasteur de Tunis, Tunisia

²University of Tunis El Manar, LR11IPT05 Laboratory of Biomedical Genomics and Oncogenetics, Institut Pasteur de Tunis, Tunisia

³University of Tunis El Manar, Faculty of Medicine of Tunis, Tunisia

⁴Department of Obstetrics and Gynecology, Aziza Othmana, University Hospital, Tunis, Tunisia

⁵Department of Urology, Charles-Nicolle University Hospital, Medical University of Tunis, Tunisia

*Corresponding author: Ahlem Amouri, Department of Histology and Cytogenetics, Institut Pasteur de Tunis 13, Place Pasteur de Tunis, Tunisia

Received: March 05, 2015; Accepted: May 10, 2015; Published: May 20, 2015

Abstract

Purpose: Male infertility is the cause in half of all childless partnerships. Numerous factors contribute to male infertility, including chromosomal aberration and Yq microdeletions. We therefore aimed to evaluate the prevalence of genetic abnormalities among non-obstructive azoospermic (NOA) Tunisian men referred for routine cytogenetic analysis to the department of cytogenetics of the Pasteur institute of Tunis.

Methods: Karyotype analyses were performed on peripheral blood lymphocytes using R-banding for 401 NOA. Molecular diagnosis of classic Yq microdeletions was performed in 90 NOA with normal karyotypes by two multiplex PCRs using six STS markers (Sequence-Tagged Site) recommended by the EAA/EMQN (European Academy of Andrology / the European Molecular Genetics Quality Network).

Results: The overall incidence of chromosomal abnormalities was 12.22% (49/401). Out of the 49 patients with abnormal cytogenetic findings, sex chromosome abnormalities were observed in 42 (85.71%) including Klinefelter syndrome in 37 (75.5%). Structure chromosome abnormalities involving autosomes (14.28%) and sex chromosomes (2.04%) were detected in 8 infertile men. Furthermore, the Yq microdeletions were seen in two patients (2.22%). Both had complete deletion of the AZFc region.

Conclusion: The occurrence of chromosome anomalies and Yq microdeletions among NOA men strongly suggests genetic testing and counseling prior to employment of assisted reproduction techniques in Tunisia.

Keywords: Male infertility; Non-obstructive azoospermia; Chromosomal abnormalities; Y-chromosome microdeletion

Introduction

Infertility is a major health problem affecting up to 15% of couples of reproductive age [1]. For many years, it was assumed that most reproductive problems could be attributed the female partner, but research in recent years has demonstrated that 30-50% of infertility is caused by male factor [2].

Until recently, there was no treatment available for men who have a complete absence of sperm in the ejaculate (azoospermia). This one accounts 10-15% of male infertility patients [3-5], among them 50-60% of which are non-obstructive azoospermia (NOA), which is characterized by the absence of sperm in the ejaculate without the obstruction of the reproductive tract pathway [6]. NOA occurs in ~1% of the male population and approximately 20-25% of NOA patients are caused by known genetic abnormalities [3, 7, 8]. Those involving chromosome anomalies to about 15%-16% [9]. The most frequent one being the 47, XXY karyotype that characterizes the Klinefelter Syndrome (KS) with a frequency 14% [9], followed by the microdeletions of the long arm of the Y chromosome (Yq) removing the azoospermia factor (AZF) region or parts with a frequency 8% [10]. However, there is still a significant proportion of NOA patients that have unknown etiology.

The establishment of in vitro fertilization using intracytoplasmic sperm injection (ICSI) as a standard treatment modality has resulted in a number of these men successfully fathering a child through surgically retrieved sperm from the testis. However, a genetic risk exists for these offspring, implying the necessity for future parents to be appropriately informed on potential consequences [11-13].

The aim of this study was to determine the prevalence of various chromosomal aberrations and the prevalence of Y-chromosome microdeletions among non-obstructive azoospermia Tunisian men attending the Pasteur Institute of Tunis.

Patients and Methods

Patients

The cytogenetic analysis has been focused on 401 Tunisian infertile patients with idiopathic non-obstructive azoospermia. These infertile men with sperm disorders were referred for karyotyping to the department of Histology and Cytogenetics at the Pasteur Institute of Tunis between January 2006 and May 2014. Among these patients, 90 non-obstructive azoospermia with normal karyotype were benefited from molecular analysis.

Patients were checked for the history of relevant medical disorders, e.g., diabetes, renal, liver disease, radiation, endocrine abnormalities (e.g., hypogonadotropic hypogonadism), exposure to toxins and/or medical affecting spermatogenesis, acquired and congenital structural defect of urogenital system, history of surgical intervention of genital tract.

Informed consent was obtained for karyotype and a molecular investigation from individual participants included in the study, and approval was given by the local ethics committee of Pasteur Institute of Tunis.

Karyotyping

Cytogenetic analysis was performed from phytohemagglutininstimulated lymphocyte cultures by routine laboratory protocol. For microscopic analysis, R-banded metaphase spreads were analyzed and abnormalities recorded according to the current International System for Human Cytogenetic Nomenclature [14]. A resolution of 550 to 700 bands per haploid karyotype was used for the routine analysis. For each patient, at least 20 well-spread metaphases were analyzed and two to five metaphases were karyotyped. When at least one of the 20 showed a loss or gain of a chromosome, especially X or Y chromosome, the number of analyzed metaphases was increased to 30. If a second abnormal cell was observed, the analysis was considered complete; otherwise, the number of metaphases was increased to 50. Sex chromosome mosaics occurring at a level of less 5% were not considered as well as pericentric inversions of chromosome 9 or other structural chromosome variants and polymorphisms that were considered as normal cytogenetic events.

Detection of AZF microdeletions by multiplex PCR

Genomic DNA was extracted from blood lymphocytes using a commercially available kit (FlexiGene Kit; Qiagen), according to the manufacturer’s instructions. Each patient was examined for six AZF loci. The STS primers used were: for AZFa sY84 and sY86, for AZFb sY127 and sY134 and for AZFc sY254 and sY255. This primer set was suggested by Simoni et al. [9] and is prescribed by the European Academy of Andrology (EAA) and European Molecular Genetics Quality Network (EMQN) [15, 16]. In addition, sY14 (STS within the SRY gene located in Yp) was tested as an internal positive control. Two multiplex PCRs were carried out in 50 μl reaction volumes containing: 200 ng of each DNA sample, 1.5mM MgCl2, 0.4 mM of each dNTP, 1.6 μM of each oligonucleotide primer: sY86, sY127, sY254 (Mix A) for the first multiplex PCR, sY84, sY134, sY255 (Mix II) for the second one, 5% dimethyl sulfoxide (DMSO) were added to 1 μl of Taq DNA polymerase. A positive control (sample from a normal fertile male), and two negative controls [(i) normal female sample, (ii) every constituent except DNA], were included in every PCR assay. The reaction mixture included thermo cycling consisted of an initial denaturation of 5 minutes to 94°C followed by 35 cycles of 30 seconds at 94°C, 30 seconds annealing at 59°°C, 30 seconds extension at 720C, finally, 7 minutes extension step at 72°C. PCR products were analyzed by electrophoresis on 3% agarose gels containing ethidium bromide and visualized by exposure to ultraviolet light. In the event of detecting deletion with a primer, the PCR assay was repeated thrice for confirmation. A STS was considered absent only after 3 amplification failures in the presence of successful amplification of internal control (SRY).

Results

Cytogenetic analysis

The average age was 38.07 ± 5.96 and the average duration of infertility was 5.26 ± 4.47 for the azoospermic patients.

Among these 401 patients, 49 cases showed abnormal karyotypes to a prevalence of 12.22%, in which 42 (10.47%) were sex chromosomal abnormalities and 7 (1.75%) were autosomal abnormalities.

The most common abnormality observed was the 47, XXY karyotype or their variant (mosaic 47, XXY/46, XY, 47, XXY/48, XXXY) consistent with KS (Figure 1), which were found in thirty seven cases (9.23%). The other chromosomal abnormalities was represented by: (6) balanced autosomal rearrangements, (2) 46, XX males (Figure 2), (2) unbalanced rearrangements, (1) 47, XYY (Figure 3), and (1) Yq deletion (Figure 4), (Table 1).

Table 1: Type and frequency of chromosomal anomalies in 401 non-obstructive azoospermia Tunisian men.




  
     
    % (No. of men with chromosomal aberration/total    No.of men karyotyped) 
  
  
     
       
       
      Non-obstructive    azoospermia men 
      12.22    % (49/401)
    Sex chromosome    aberrations
    Autosomal chromosome    aberrations
  
  
    Karyotypes
    %
    Karyotypes
    %
  
  
    47,    XXY
      Mosaic    47, XXY:
      *46,    XY/47, XXY
      *47,    XXY (85%)/48, XXXY (15%)
      47,    XYY
      45,    X (15%)/46, XY (85%)
      46,    X, del (Y) (q11.2 qter)
      XX    males (46, XX)
       
    8.23    (33/401)
      1    (4/401)
      (3)
      (1)
      0.25    (1/401)
      0.25    (1/401)
      0.25    (1/401)
      0.5    (2/401)
       
    Reciprocal    translocation :
      *46,    XY, t (9 ; 22) (q11;p11)
      *46,    XY, t (7;16) (p11;p13)
      *46,    XY, t (4;6) (p12;p22)
      *46,    XY, t (4;17) (q11;p11)
      Robertsonian    translocation :
      *45,    XY, der (13;14) (q10;q10)
      Inversion:
      *46,    XY, inv (7) (q22;q35)
      Supernumerary marker    chromosomes:
      *    46, XY/47, XY, +mar
    1    (4/401)
      (1)
      (1)
      (1)
      (1)
      0.25    (1/401)
      (1)
      0.25    (1/401)
      (1)
      0.25    (1/401)
      (1)
  
  
     
    Subtotal
    10.47 (42/401)
    Subtotal
    1.75 (7/401)



Table 1:  Type and frequency of chromosomal anomalies in 401 non-obstructive azoospermia Tunisian men.

Figure 1: Karyotype of a patient with Klinefelter syndrome: 47, XXY.

    
    
    Figure 1:  Karyotype of a patient with Klinefelter syndrome: 47, XXY.

Figure 2: Karyotype of a patient with 47, XYY.

    
    
    Figure 2:  Karyotype of a patient with 47, XYY.

Figure 3: Karyotype of a patient with male syndrome 46, XX.

    
    
    Figure 3:  Karyotype of a patient with male syndrome 46, XX.

Figure 4: Karyotype of a patient with 46,X,del (Y)(q11.2 qter).

    
    
    Figure 4:  Karyotype of a patient with 46,X,del (Y)(q11.2 qter).

Of balanced rearrangements are identified in six patients including: four with balanced reciprocal translocations, one with balanced Robertsonian translocations between chromosome 13 and chromosome 14 (Figure 9), and one paracentric inversion in chromosome 7 (Figure 10). An unbalanced rearrangement was identified in the two patients: one patient with supernumerary marker chromosome in a low level mosaic [3 out 20 metaphases] (Figure 11), and the last one had three cells with a 45, X constitution.

Figure 9: Karyotype of a patient with Robertsonian translocation: 45, XY, der(13;14)(q10;q10).

    
    
    Figure 9:  Karyotype of a patient with Robertsonian translocation: 45, XY,
der(13;14)(q10;q10).

Figure 10: Karyotype of a patient with inversion paracentric of chromosome 7: 46, XY, inv(7)(q22;q35).

    
    
    Figure 10:  Karyotype of a patient with inversion paracentric of chromosome
7: 46, XY, inv(7)(q22;q35).

Figure 11: Karyotype of a patient with small supernumerary chromosome: 47, XY, +mar.

    
    
    Figure 11:  Karyotype of a patient with small supernumerary chromosome:
47, XY, +mar.

Yq chromosome microdeletion screening

Screening for AZF microdeletions was carried out in the 90 NOA men with normal karyotype. Using the EAA/EMQN criteria, AZF region microdeletions on the Y chromosome were found in patients with a frequency of 2.22%. The failed amplification of sY254 and sY255 markers indicates a complete deletion in the AZF sub-region (Figure 12).

Figure 12: Gel electrophoresis analysis of two multiplex PCRs (PCR A and B) showing deletion in the AZF region of the Y chromosome. (P1, P2 & P3) three NOA patient with normal karyotype. (M) size marker (100bp ladder), (+) fertile man sample, (-) female sample, (EA and EB) water. Patient (P2) have an absence of the sY254 and sY255 markers, indicating a complete deletion in the AZFc subregion.

    
    
    Figure 12:  Gel electrophoresis analysis of two multiplex PCRs (PCR A and
B) showing deletion in the AZF region of the Y chromosome.
(P1, P2 & P3) three NOA patient with normal karyotype. (M) size marker
(100bp ladder), (+) fertile man sample, (-) female sample, (EA and EB) water.
Patient (P2) have an absence of the sY254 and sY255 markers, indicating a
complete deletion in the AZFc subregion.

Discussion

Chromosomal abnormalities have emerged as one of the major genetic factors contributing to male infertility. In this study, the prevalence of major chromosomal anomalies was 12.22% in NOA with primary infertility. This was lower than in previous studies of Tunisian population 14.10 to 23.62%, but the incidence in other populations has been found to be between 5.43% and 19.44% (Table 2).

Table 2: Comparison of chromosomal anomalies between this study and other similar studies.




  
    Authors 
    Region 
    Years 
    Prevalence of chromosomal aberration (No.of    cases /Total No;) 
  
  
    Tuerlings et    al. [39] 
    Netherlands
    1998
    6.45% (4/62)
  
  
    Nagvenkar et    al. [34] 
    India
    2005
    14.29% (6/42)
  
  
    Elghazel et    al.[58] 
    Tunisia
    2006
    23.62% (81/343)
  
  
    Mohammed et    al. [59] 
    Kuwait
    2007
    19.44% (21/108)
  
  
    Ng et al. [60] 
    Hong Kong
    2009
    21.1%   (5/71)
  
  
    Kosar et    al. [61] 
    South of Turkey
    2010
    5.43%   (5/92)
  
  
    Mafra et    al. [62] 
    Brazil
    2011
    11.62% (5/43)
  
  
    Ghorbel et    al. [48] 
    Tunisia
    2012
    22.22% (12/54)
  
  
    Zhang et    al. [63] 
    Northeast China
    2012
    17.28% (14/81)
  
  
    Cavkaytar et    al. [64] 
    Turkey
    2012
    11.22% (22/196)
  
  
    Al-Achkar et    al. [65] 
    Syria
    2013
    17.55% (17/97)
  
  
    Amouri et    al. [66] 
    Tunisia
    2014
    14.10% (46/328)
  
  
    This study
    Tunisia
    2015
    12.22% (49/401)



Table 2:  Comparison of chromosomal anomalies between this study and other
similar studies.

• Sex chromosome abnormalities

Sex chromosome abnormalities are the most frequent chromosome related cause of infertility. In our study, we have found thirty three men (8.23%) with 47, XXY karyotype. A mosaic 46, XY/47, XXY/ 48, XXXY karyotype was found in four azoospermic male (1%). Clinically, these abnormalities are associated with severe spermatogenic failure causing a marked reduction in testicular size which can be associated with gynecomastia, and/or a reduction of pubic and facial and/or hair. And biologically, it is associated with a form of primary hypogonadism. Fluorescence in situ hybridization (FISH) analysis has demonstrated that the frequency of aneuploidy for the sex chromosomes varies from 2% [17] to 45% [18] in the sperm of men who appear to have a non-mosaic KS, and 1.5% [19] to 7% [20] in sperm from mosaics KS. The majority of babies born to men with KS have been normal although chromosomally abnormal fetuses have been reported [21-23] studied embryos by preimplantation genetic diagnosis (PGD) and reported a significant fall in the rate of normal embryos (54%) from KS patients when compared with the controls (72%). Even that there appears to be a small increased risk for these men, it is advised that PGD or prenatal diagnosis be performed before ICSI to ensure that the offspring is not aneuploid [24].

Besides, the second most predominant constituent is represented by the 46, XX, which it has been identified in 2 cases (0.5%). This rare condition was initially named “XX male syndrome”. However, this was revised in October 2005 to its current nomenclature of “46, XX testicular disorder of sex development” (DSD) [25, 26]. This disorder has been reported with an incidence of 0.9% in azoospermic males [27]. Phenotypically the adults are similar to patients with KS. However, 46, XX DSD are shorter, and in some cases they have genital abnormalities [28]. The treatment of fertility of these patients can only be handled using the artificial insemination with donation sperm. Moreover, the donor sperm is banned in Tunisia and in the rest of the Sunni Islamic world [29] and the only possibility paternity for these patients remain the adoption.

The 47, XYY karyotype was seen in one patient. Men with the extra Y chromosome are mostly fertile, but azoospermia may be seen in some cases [30-32].

We also observed in our samples, two others gonosomes aberrations related with the Y chromosome aberration: one case with long arm deletions in all cell, and the second one with the 45X/XY mosaicism constituent. Effectively, loss of genes on the Yq, which plays a primary role in the regulation of different stages of spermatogenesis, is particularly dramatic in spermatozoa production [33].

• Autosomal chromosome abnormalities

The prevalence of autosomal abnormalities in our cohort of NOA was 1.75% (7/401), represented by; six cases with balanced rearrangements [(4) reciprocal translocation, (1) Robertsonian translocation and (1) inversion] and one case with unbalanced rearrangement [(1) small supernumerary marker chromosome (sSMC)].

An association between balanced autosomal translocation and infertility has been reported among NOA men [34-37]. In our samples, the reciprocal translocation were seen four cases involving these translocation [t(9;22); t(4;6); t(7;16); t(4;17)] (Figure 5-8). Otherwise, one case had Robertsonian translocation involving chromosome 13 and 14. Most translocations have no effect on other tissues but can severely impair spermatogenesis [38]. Using the ICSI in this group may increase the inheritance of paternal genetic disorder to offspring due to disrupted meiotic pairing and segregation [38, 39]. Indeed, according to the translocations carried the percentage of unbalanced gametes varies between 2.7% to 26.5% [40]. Both the chromosomes involved in translocation and the location of the breakpoints are likely to be determining factors for the fertility status of the patient. In addition, Robertsonian translocation can result in offspring with Down syndrome or Patau’s syndrome or in gestational loss of concepts with monosomy of chromosome 13, 14 or 21, or trisomy of chromosome 14, which are lethal [32]. Preimplantation genetic diagnosis (PDG) by FISH (Fluorescent in Situ Hybridation) is recommended for the autosomal chromosome aberrations, in order to have an estimation of the risk of abnormal offspring and to adopt genetic counselling which accordingly may be useful for couples who opt for ART.

	% (No. of men with chromosomal aberration/total No.of men karyotyped)
Non-obstructive azoospermia men 12.22 % (49/401)	Sex chromosome aberrations		Autosomal chromosome aberrations
	Karyotypes	%	Karyotypes	%
	47, XXY Mosaic 47, XXY: 46, XY/47, XXY 47, XXY (85%)/48, XXXY (15%) 47, XYY 45, X (15%)/46, XY (85%) 46, X, del (Y) (q11.2 qter) XX males (46, XX)	8.23 (33/401) 1 (4/401) (3) (1) 0.25 (1/401) 0.25 (1/401) 0.25 (1/401) 0.5 (2/401)	Reciprocal translocation : 46, XY, t (9 ; 22) (q11;p11) 46, XY, t (7;16) (p11;p13) 46, XY, t (4;6) (p12;p22) 46, XY, t (4;17) (q11;p11) Robertsonian translocation : 45, XY, der (13;14) (q10;q10) Inversion:* 46, XY, inv (7) (q22;q35) Supernumerary marker chromosomes: 46, XY/47, XY, +mar	1 (4/401) (1) (1) (1) (1) 0.25 (1/401) (1) 0.25 (1/401) (1) 0.25 (1/401) (1)
	Subtotal	10.47 (42/401)	Subtotal	1.75 (7/401)

Authors	Region	Years	Prevalence of chromosomal aberration (No.of cases /Total No;)
*Tuerlings et al.* [39]	Netherlands	1998	6.45% (4/62)
*Nagvenkar et al.* [34]	India	2005	14.29% (6/42)
*Elghazel et al.[58]*	Tunisia	2006	23.62% (81/343)
*Mohammed et al.* [59]	Kuwait	2007	19.44% (21/108)
*Ng et al.* [60]	Hong Kong	2009	21.1% (5/71)
*Kosar et al.* [61]	South of Turkey	2010	5.43% (5/92)
*Mafra et al.* [62]	Brazil	2011	11.62% (5/43)
*Ghorbel et al.* [48]	Tunisia	2012	22.22% (12/54)
*Zhang et al.* [63]	Northeast China	2012	17.28% (14/81)
*Cavkaytar et al.* [64]	Turkey	2012	11.22% (22/196)
*Al-Achkar et al.* [65]	Syria	2013	17.55% (17/97)
*Amouri et al.* [66]	Tunisia	2014	14.10% (46/328)
This study	Tunisia	2015	12.22% (49/401)

Authors	Region	Years	Prevalence of Yq microdeletions (No. of cases /Total)	Karyotype
*Arruda et al.* [55]	Brasilia	2007	43.48% (10/23)	46, XY
*Elhawary et al.[67]*	Egypt	2010	39.28% (11/28)	46, XY
*Behulova et al.[53]*	Slovakia	2011	3.35% (8/226)	46, XY
*Sun et al.* [54]	China (Shanghai)	2012	9.32% (33/354)	46, XY
*Wettasinghe et al.* [52]	Sri Lanka	2012	1.96% (3/153)	46, XY
*Saliminejad et al.[50]*	Iran	2012	2.13% (2/94)	46, XY
*Chellat et al.* [51]	Alegria	2013	2.04% (2/49)	NA
*Hammami et al.* [68]	Tunisia	2014	2.70% (2/74)	46, XY
This study	Tunisia	2015	2.22% (2/90)	46, XY
NA= not available;

Genetic Diagnosis in Non-Obstructive Azoospermic Tunisian Men

Abstract

Introduction

Patients and Methods

Patients

Karyotyping

Detection of AZF microdeletions by multiplex PCR

Results

Cytogenetic analysis

Yq chromosome microdeletion screening

Discussion

Discussion

Molecular analysis

Conclusion

Acknowledgment

References